The metabolic pathways and the biogenesis of peroxisomes are linked to at least 17, often fatal, human peroxisomal disorders. Genetic strategies in multiple model systems have systematically uncovered 29 PEX genes, encoding proteins called peroxins. Surprisingly, homologues of very few of these proteins are used in protein transport to other organelles, and many features of peroxisome biogenesis are unique. Remarkably, most human PEX genes were defined based on their counterparts in other organisms, particularly yeasts. However, although the molecular basis of most human peroxisomal biogenesis disorders is known, we still do not comprehend the molecular functions of many peroxins. Peroxisomal proteins are targeted to the matrix by PTS1 and PTS2 sequences, while membrane proteins (PMPs) reach their destination via the use of mPTSs. After their synthesis in the cytosol, cargoes with PTS1 and PTS2 sequences are recognized by receptors, Pex5 and Pex7, respectively. The receptor-cargo complexes are delivered to a docking subcomplex on the peroxisome membrane, which transiently associates with a RING subcomplex, to form the importomer that includes the translocon. During import, both Pex5 and Pex7 are believed to enter (partly or completely) the peroxisome lumen, release cargo and recycle to the cytosol. While much is known regarding the docking of receptor-cargo complexes on the cytosolic face of the peroxisome membrane, the terminal steps (in the translocon or the peroxisome lumen), such as cargo- and receptor-release, are not well defined. Multiple peroxin interactions have been deduced, but very few macromolecular complexes have been purified, characterized and reconstituted functionally. In contrast to the targeting of matrix proteins, many critical issues remain regarding PMP biogenesis. Finally, there appears to be a novel, but poorlyunderstood, role for ubiquitination in peroxisome biogenesis. The aims (see below) are to use P. pastoris to substantially advance our knowledge of the actions of molecular machines used for peroxisome biogenesis. 1. Purify and functionally reconstitute protein complexes involved in peroxisomal matrix protein import. 2. Define the targeting mechanism of different classes of peroxisomal membrane proteins (PMPs). 3. Understand the mechanism of peroxisomal targeting, protein-protein interactions and the function of Pex8, the only intraperoxisomal peroxin necessary for matrix protein import. 4. Elucidate further the role of ubiquitination in peroxisome biogenesis.